Human osteoclasts/osteoblasts 3D dynamic co­culture system to study the beneficial effects of glucosamine on bone microenvironment.

Glucosamine (GlcN) functions as a building block of the cartilage matrix, and its multifaceted roles in promoting joint health have been extensively investigated. However, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. As a result, the benefit of GlcN application in bone disorders remains controversial. In order to further elucidate the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, the effect of GlcN treatment was investigated in human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were cultured with two­dimensional (2D) traditional methods or co­cultured in a 3D dynamic system more closely resembling the in vivo bone microenvironment. Under these conditions, osteoclastogenesis was supported by hOBs and sizeable self­assembling aggregates were obtained. The differentiated hOCs were evaluated using tartrate­resistant acid phosphatase assays and osteogenic differentiation was monitored by analyzing mineral matrix deposition via Alizarin Red staining, with expression of specific osteogenic markers determined via reverse transcription­quantitative PCR. It was found that crystalline GlcN sulfate was effective in decreasing osteoclastic cell differentiation and function. hOCs isolated from patients with OA were more sensitive compared with those from healthy donors. Additionally, GlcN exhibited anabolic effects on hOCs both in 2D conventional cell culture and in hOC/hOB 3D dynamic co­culture. The present study demonstrated for the first time the effectiveness of a 3D dynamic co­culture system for characterizing the spectrum of action of GlcN on the bone microenvironment, which may pave the way for more fully determining the potential applications of a compound such as GlcN, which is positioned between pharmaceuticals and nutraceuticals. Based on the present findings, it is hypothesized that GlcN may have potential benefits in the treatment of osteopenic diseases such as osteoporosis, as well as in bone maintenance.

Anatase-based implants nanocoating on stem cells derived from adipose tissue.

PURPOSE: The aim of this study was to investigate the effect of a new anatase coating with antibacterial properties (Bactercline anatase coating [BAC]) on dental implants in the commitment of stem cells derived from adipose tissue to osteoblasts. MATERIALS AND METHODS: Using real-time reverse transcription polymerase chain reaction, the quantitative expression of specific genes, such as transcriptional factors (runx2 and sp7), bone-related genes (spp1, col1a1, col3a1, alpl, and fosl1), and mesenchymal stem cells marker (eng), was examined. RESULTS: BAC caused induction of bone-related genes such as sp7, fosl1, alpl, and spp1. In contrast, the expression of runx2, col3a1, and col1a1 was decreased in stem cells treated with BAC with respect to untreated cells. CONCLUSION: The obtained results are relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Prion proteins (PRNP and PRND) are over-expressed in osteosarcoma.

Although osteosarcoma is the most common bone malignancy, the molecular and cellular mechanisms influencing its pathogenesis have remained elusive. Prion proteins (PRNP and PRND), known mostly for its involvement in neurodegenerative spongiform encephalopathies, have been recently demonstrated to be involved in resistance to apoptosis, tumorigenesis, proliferation, and metastasis. The main aim of research was to study whether prion proteins were over-expressed in human osteosarcoma, and if prion proteins could have a role also in osteosarcomas. We evaluated differential gene expression between 22 cases of osteosarcoma and 40 cases of normal bone specimens through cDNA microarray analysis spanning a substantial fraction of the human genome. PRNP and PRND are significantly over-expressed in osteosarcoma. PRNP and PRND appear involved with some important genes related to tumorigenesis and apoptosis. PRNP is linked to PTK2, RBBP9, and TGFB1 while PRND is linked to TNFSF10, BCL2A1, NFKB2, and TP53RK. Increased expression on Affymetrix arrays of prion proteins seems to be associated with the development of osteosarcoma. Prions seem to induce a negative regulation of apoptosis, thus promoting osteosarcoma development and progression. Osteosarcoma is a very aggressive tumor and even after modern chemotherapy and excision of tumors efforts are needed to improve clinical outcome. Since Prion proteins seem to be related to osteosarcoma development, their inhibition could represent a new approach to the molecular treatment of osteosarcoma.

Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: a review.

Injection of hyaluronan into osteoarthritic joints restores the viscoelasticity, augments the flow of joint fluid, normalizes endogenous hyaluronan synthesis, and improves joint function. Chitosan easily forms polyelectrolyte complexes with hyaluronan and chondroitin sulfate. Synergy of chitosan with hyaluronan develops enhanced performances in regenerating hyaline cartilage, typical results being structural integrity of the hyaline-like neocartilage, and reconstitution of the subchondral bone, with positive cartilage staining for collagen-II and GAG in the treated sites. Chitosan qualifies for the preparation of scaffolds intended for the regeneration of cartilage: it yields mesoporous cryogels; it provides a friendly environment for chondrocytes to propagate, produce typical ECM, and assume the convenient phenotype; it is a good carrier for growth factors; it inactivates metalloproteinases thus preventing collagen degradation; it is suitable for the induction of the chondrogenic differentiation of mesenchymal stem cells; it is a potent means for hemostasis and platelet delivery.

Evaluation of gene expression in MG63 human osteoblastlike cells exposed to tantalum powder by microarray technology.

Conventional orthopedic implants are composed from titanium. To improve some characteristics (ie, volumetric porosity, modulus of elasticity, frictional modulus), a new porous tantalum biomaterial has been developed and its biocompatibility reported. By using DNA microarrays containing 20,000 genes, several genes whose expression were significantly up- or down-regulated were identified in an osteoblastlike cell line (MG63) cultured with tantalum powder (TP). The differentially expressed genes cover a broad range of functional activities: signaling transduction; transcription; cell cycle regulation, proliferation, and apoptosis; and cytoskeleton formation. To the authors' knowledge, the data reported represent the first genetic portrait of TP.

Effectiveness of fresh frozen and cryopreserved homologue iliac crest grafts used in sinus lifting: a comparative study.

In the last decade, several investigators have reported that autologous and homologous fresh frozen bones (FFB) are effective materials to restore alveolar ridges previous to insert dental implants. Recently we have used cryopreserved homologue grafts (CFFB). Here we reported a retrospective comparative study between implants inserted in FFB and CFFB evaluate their clinical outcome. Patients were treated with a split mouth scheme for bone grafting with FFB and CFFB and spiral family implants (SPI) were inserted in the same surgical time. Several variables (patient, grafts, anatomic site, implant, prosthetic restoration) were investigated. Implant' failure and peri-implant bone resorption were considered as predictor of clinical outcome. A total of 84 SFIs were inserted in 12 patients. Implants were inserted to replace 8 incisors, 4 cuspids, 31 premolars and 41 molars. The mean follow-up was 14 months. Three out of 84 implants was lost (i.e. survival rate SVR = 96.4%) and no differences were detected among the studied variables. Similar result was obtained by analyzing the crestal bone resorption around implant' neck (i.e. success rate). FFB and CFFB have high and comparable survival and success rate. Implants inserted with one step surgical procedure in native (i.e. not grafted) bone, FFB and CFFB have similar clinical outcome.

Osteoplant acts on stem cells derived from peripheral blood.

OBJECTIVES: The osteoplant is an equine, flexible, heterologous, deantigenic, cortical, and spongy bone tissue, totally reabsorbable, used for implantation of bone tissue, to restore skeletal, even weight-bearing structures. However, how the osteoplant alters osteoblast activity to promote bone formation is poorly understood. MATERIALS AND METHODS: To study how the osteoplant induces osteoblast differentiation in mesenchymal stem cells, the expression levels of bone-related genes, and mesenchymal stem cell markers are analyzed, using real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RESULTS: The osteoplant causes induction of osteoblast transcriptional factors such as osterix (RUNX2), and of bone-related genes such as osteopontin (SPP1) and osteocalcin (BGLAP). In contrast the expression of ENG (CD105) is significantly decreased in stem cells treated with osteoplant, with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. CONCLUSION: The obtained results can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Effects of pulsed electromagnetic fields on human osteoblastlike cells (MG-63): a pilot study.

BACKGROUND: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes. QUESTIONS/PURPOSES: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures. METHODS: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome. RESULTS: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). CONCLUSIONS AND CLINICAL RELEVANCE: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.

Reconstruction of severely atrophic jaws using homografts and simultaneous implant placement: a retrospective study.

In the past decade, several investigators have reported that implants inserted in autografts in the same operation (ie, simultaneously inserted implants [SIIs]) have achieved excellent results. However, no report regarding SIIs placed in fresh frozen bone (FFB) is available. Thus, the authors planned a retrospective study on a series of SIIs placed in homologue FFB (but not immediately loaded) to evaluate their clinical outcome. In addition, a comparison with implants inserted in FFB in a second stage (ie, delayed inserted implants) was performed. Seventeen patients were grafted with FFB, and 48 implants were inserted in the same operation. Implant diameter and length ranged from 3.25 to 4.0 mm and from 10.0 to 15 mm, respectively. Data were compared with 302 implants inserted in FFB in a second operation during the same period in 64 patients. Analyzing SIIs, it was noted that only 3 implants were lost (ie, survival rate [SVR] = 93.7%), and no differences were detected among the studied variables by using lost implants as a predictor of clinical outcome. On the contrary, by using crestal bone resorption around the implant's neck and specific cutoff values, it was possible to demonstrate that prosthetic restoration (ie, removable overdentures) correlated with a statistically significant lower delta insertion abutment junction (ie, reduced crestal bone loss) and thus with a better clinical outcome. By comparing SIIs with implants inserted in a second stage in FFB, a better outcome for delayed implants was demonstrated. Implants inserted simultaneously with FFB grafts had a high survival and success rate. SIIs inserted in FFB can be considered reliable devices, although a higher marginal bone loss is to be expected when fixed prosthetic restorations are used. Implants inserted in a second surgical stage have a better SVR and success rate than SIIs.

Engipore acts on human bone marrow stem cells.

OBJECTIVES: Porous HA scaffolds are promising materials for tissue engineering because they offer a tridimensional support and serve as template for cell proliferation and at last tissue formation. Engipore provide a natural 3D scaffold with organic fibrous material in bone. However, how this material alters osteoblast activity to promote bone formation is poorly understood. MATERIALS AND METHODS: To study how Engipore can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed. RESULTS: Engipore causes a significant induction of osteoblast transcriptional factors like SP7 and RUNX2 and of the bone-related gene osteocalcin (BGLAP). The expression of CD105 was not significantly changed in stem cells treated with Engipore with respect to untreated cells, while SSP1 (osteopontin) was significantly down expressed thus reducing osteoclast activity. CONCLUSIONS: The obtained results can be relevant to better understand the molecular mechanism of bone regeneration.

Early effects of p-15 on human bone marrow stem cells.

OBJECTIVES: Peptide-15 (P-15) is an analogue of the cell binding domain of collagen. P-15 has been shown to facilitate physiological to process in a way similar to collagen, to serve as anchorage for cells, and to promote the binding, migration and differentiation of cells. However, how P-15 alters osteoblast activity to promote bone formation is poorly understood. To study the osteoinductive properties of peptide P-15, we analyzed the expression levels of bone related genes in human mesenchymal stem cells treated with this biomaterial. MATERIAL AND METHODS: Using real time Reverse Transcription-Polymerase Chain Reaction the quantitative expression of specific genes, like transcriptional factors (RUNX2 and SP7), bone related genes (SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1) and mesenchymal stem cells marker (ENG) were examined. RESULTS: P-15 causes a considerable induction of osteoblast transcriptional factor like osterix (SP7) and of the bone related genes osteopontin (SPP1) and osteocalcin (BGLAP). In contrast the expression of endoglin (ENG) was markedly decreased in stem cells treated with P-15 respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. CONCLUSIONS: The present study shows the effect of P-15 on mesenchymal stem cells in the early differentiation stages: P-15 is an inducer of osteogenesis on human stem cells as indicated by the activation of bone related markers SP7, SPP1 and BGLAP.The results may allow a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Bio-Oss®acts on Stem cells derived from Peripheral Blood.

OBJECTIVES: This study aims to study how Bio-Oss® can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells markers using real time Reverse Transcription-Polymerase Chain Reaction. METHODS: PB-hMSCs stem preparations were obtained for gradient centrifugation from peripheral blood of healthy anonymous volunteers, using the Acuspin System-Histopaque 1077. The samples were then cultured for 7 days for RNA processing, and the expression was quantified using real time PCR. RESULTS: Bio-Oss® caused an induction of osteoblast transcriptional factor like RUNX2 and of bone related genes; SPP1 and FOSL1. In contrast, the expression of ENG was significantly decreased in stem cells treated with Bio-Oss® with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. CONCLUSION: The results obtained can be relevant to enhance the understanding of the molecular mechanism of bone regeneration and can act as a model for comparing other materials with similar clinical effects.

PerioGlas® Acts on Human Stem Cells Isolated from Peripheral Blood.

BACKGROUND: PerioGlas® (PG) is an alloplastic material used for grafting periodontal osseous defects since 1995. In animal models, it has been proven that PG achieves histologically good repair of sur-gically created defects. In clinical trials, PG was effective as an adjunct to conventional surgery in the treatment of intrabony defects. Because the molecular events due to PG that are able to alter osteob-last activity to promote bone formation are poorly understood, we investigated the expression of os-teoblastic related genes in mesenchymal stem cells exposed to PG. METHODS: The expression levels of bone related genes like RUNX2, SP7, SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1 and mesenchymal stem cells marker (CD105) were analyzed, using real time reverse transcription-polymerase chain reaction. Pearson's chi-square (χ(2)) test was used to detect markers with significant differences in gene expression. RESULTS: PG caused induction of osteoblast transcriptional factor (like RUNX2), bone related genes osteopontin (SPP1), osteocalcin (BGLAP) and alkaline phosphatase (ALPL). All had statistical sig-nificant P values (< 0.05). CONCLUSION: PG has a differentiation effect on mesenchymal stem cells derived from peripheral blood. The obtained results can be relevant to better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Pulsed electromagnetic fields and low intensity pulsed ultrasound in bone tissue.

The use of physical stimuli to modulate osteogenetic response and favour fracture healing has been the subject of research for many years now. Currently, 78% of hospitals in the USA provide this treatment at 3 months from the trauma.In literature, the findings of many clinical studies agree in confirming that biophysical stimuli are able to lead to healing in 75-85% of patients with nonunions. Prospective, randomized and double-blind studies show that by employing biophysical stimuli the time needed for a fresh fracture to heal can be reduced "on average" by 25-38%. The treatment is suggested for healing of fresh fractures that are characterized as "risk fractures", fractures that can evolve in nonunions and that amount to 20% of all the fractures; this justifies the use of biophysical stimuli, with a favourable cost-benefit ratio.Current orthopaedics reviews the different modalities of biophysical treatment in search of solutions most adequate to the pathology, the characteristics of the fracture and those of the patient. It is up to the orthopaedist to assess whether the biomechanical conditions of stability of the fracture site are such as not to jeopardize the osteogenetic process. International clinical experience shows that success in biophysical therapy for bone regeneration depends on certain principal factors: suitable indication, efficacy of the device employed, method of stimulation and - of crucial importance - patient compliance. If these principles are kept in mind, the percentage of success of union, obtained with biophysical stimulation, exceeds 90%.

Anatase nanosurface regulates microRNAs.

Titanium is the criterion standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. There are 3 allotropic forms of titanium dioxide: brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biologic effects and specifically induce bone formation. To get more information as regards the osteogenic effect of AC, we used microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts exposed to AC. Transduction, transcription, and translation are the 3 levels of regulation of cell activity. Recently, a new type of translation regulation has been identified: RNA interference. RNA interference is a process in which miRNA (i.e., noncoding RNAs of 19-23 nucleotides) can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes a few hundred miRNAs that can posttranscriptionally repress thousands of genes. miRNA oligonucleotide microarray provides a novel method of carrying out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified in osteoblast-like cell line (MG-63) cultured with AC several miRNA whose expression had been significantly modified. The data reported constitute, to our knowledge, the first study on translation regulation in osteoblasts exposed to AC. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Exclusion of COL2A1 and VDR as developmental dysplasia of the hip genes.

Developmental dysplasia of the hip (DDH) is a spectrum of disorders affecting the proximal femur and/or acetabulum leading to an abnormal formation of the hip. Genetic factors are involved in the etiology of DDH. Early recognition of DDH affords the best results from treatment and a better knowledge of the genetics of DDH could enhance early diagnosis. Variants in the Type II collagen (COL2A1) and vitamin D receptor (VDR) genes have been associated with patients with osteoarthritis of the hip secondary to DDH, suggesting these genes could contribute to DDH. To see whether there was linkage between the COL2A1/VDR locus and nonsyndromic DDH, we conducted a linkage study on 11 families with multiple cases of DDH. We demonstrated no evidence of linkage between the COL2A1/VDR locus and nonsyndromic DDH (LOD score < -2), suggesting, although variants in these genes could play a role in osteoarthritis in patients with DDH, they do not contribute to nonsyndromic DDH. The search for causal gene variants should proceed with other candidates.

Zirconium oxide coating improves implant osseointegration in vivo.

OBJECTIVES: Zirconium is widely used as material for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, zirconium becomes zirconium oxide (ZO, chemically ZrO(2)) which is biocompatible. ZO can be also prepared as a colloidal suspension and then used to coat surfaces. Zirconium oxide coating (ZOC) can potentially have specific biologic effects. METHODS: The effect of ZOC on bone throughout an in vivo study using dental implants covered with ZOC and then inserted in rabbit tibia was tested in this study. RESULTS: The histologic analysis demonstrated that (1) bone growth is more evident around ZOC fixtures than in controls and (2) a more mature bone is present in the peri-implant ZOC surface than in controls. SIGNIFICANCE: ZOC can enhance implant osseointegration.

Genetic effect of anatase on osteoblast-like cells.

Titanium is the gold standard among materials used for prosthetic devices, because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. Three allotropic forms of titanium dioxide exist, that is brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biological effects. Here we are testing the effect of AC on osteoblast-like cells (MG63) by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to AC. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured on AC, several genes whose expression was significantly up- or downregulated. They cover a broad range of functional activities: signaling transduction, immunity, cell cycle regulation, lysosomes composition and vesicular transport, cell adhesion, cytoskeleton and extracellular matrix components, proliferation, and apoptosis. The data reported constitute, to our knowledge, the first genetic portrait of AC effects. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Genetic effect of zirconium oxide coating on osteoblast-like cells.

Zirconium is widely used as material for prosthetic devices because its good mechanical and chemical properties. When exposed to oxygen, zirconium becomes zirconium oxide (ZrO(2)), which is biocompatible. ZrO(2) can be also prepared as a colloidal suspension and then used to coat surfaces. Zirconium oxide coating (ZrO(2)C) can potentially have specific biologic effects, and among them is bone formation related to implant osseointegration. How this biomaterial alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to ZrO(2)C. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured with ZrO(2)C several genes whose expression was significantly upregulated or downregulated. The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) signal transduction, (c) immunity, and (d) cytoskeleton component. The data reported are, to our knowledge, the first genetic portrait of ZrO(2)C effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Anatase coating improves implant osseointegration in vivo.

Titanium is the gold standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. There are three allotropic forms of titanium dioxide: brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biologic effects. We test the effect of AC on bone throughout an in vivo study by using dental implants covered with AC and then inserted in rabbit tibia. The histologic analysis has demonstrated that 1) bone growth is more evident around AC fixtures than in controls and 2) a more mature bone is present in the periimplant AC surface than in controls. Therefore, we have demonstrated that AC enhances implant osseointegration.